First Report of Southern Blight on Polygonatum sibiricum Caused by Sclerotium delphinii in China

Polygonatum sibiricum Delar. ex Redoute is a plant species used for medicine and food. On one hand, its rhizomes have potential medicinal values such as enhancing immunity, anti-aging, anti-tumor and antibacterial as well as the effects of improving memory and reducing blood lipid and sugar. On the other hand, the rhizomes can also be used as raw materials for drinks, preserves, and health products (Su et al. 2018). The annual demand of P. sibiricum is about 3500-4000 tons in China, and the market demands and the price continue to rise in recent years (Su et al. 2018). In August 2019, there was an outbreak of southern blight in the P. sibiricum planting fields (N30°04'06″, E115°39'47″) of Luotian County in Hubei province of China. Approximately 30% of plants were affected in many fields (333.33 ha). We observed that the surface of the infected rhizome and the surrounding soils were covered with white hyphae and sclerotia. The hyphae gradually extended downward to the rhizomes, causing rhizome rot and leaf yellowing and wilting. Mycelial fragments and sclerotia from ten symptomatic rhizomes were collected in the fields and incubated directly on potato dextrose agar (PDA containing 50 µg/ml kanamycin) at 27℃. The fungal colonies were transferred to PDA after two days of cultivation. The white colonies were formed with fluffy aerial mycelia, which grew radially with an average growth rate of 20.54±0.52 mm/d (n=10). The color of the sclerotia was milky white at first, and then gradually turned to beige and yellow-brown. After two-week-incubation, the sclerotia became dark brown. Most of the sclerotia were spherical or nearly spherical, with round-bulges on the surface. The number of mature sclerotia produced per plate ranged from 8-23 (n=10), and the size ranged from 2.5×3.0 mm to 7.5×13.0 mm (5.95 ± 2.34×7.51 ± 2.88 mm; n=50). In addition, clamp connections were observed under the microscope. For molecular identification, genomic DNA was extracted from isolate HJ-1 using the CTAB method (Mahadevakumar et al. 2018). The internal transcribed spacer (ITS) regions of rDNA were amplified with the primers ITS1/ITS4 (White et al. 1990). The resulting showed ITS sequence (Accession number: MW049362) was 99.66% homology with Sclerotium delphinii according to the GenBank database. In addition, the second largest subunit of RNA polymerase II gene (RBP2) and part of the elongation factor 1-alpha (EF1-α) gene were amplified by using the primers RPB26F/RPB2-7CR (Liu et al. 1999) and EF595F/EF1160R, respectively (Wendland and Kothe 1997). RPB2 gene sequence was deposited in GenBank (Accession number: MW415935), and was 99.53% similarity identity to Athelia rolfsii isolate MSB5-1. TEF-1α sequence was deposited in GenBank (Accession number: MW415934), and was 91.35% similarity to S. delphinii strain Sd_405. Because there are very few reference sequences of RPB2 genes from S. delphinii in GenBank to compare, we choose the ITS and TEF-1α gene sequences to construct the concatenated phylogenetic tree by the neighbor-joining method (Tamura et al. 2013). The results showed that HJ-1 was clustered with S. delphinii isolates selected from NCBI database. Based on morphological and molecular characteristics, the fungus was identified as S. delphinii Welch (teleomorph Athelia rolfsii (Curzi) C.C. Tu & Kimbr). Pathogenicity tests were performed on the healthy leaves, roots, stems and plants (n=3) of P. sibiricum. Each sample was inoculated with one sclerotia produced from a fifteen-day-old colony and there was on wound treatment. These inoculated and control samples (treated with sterile water) were incubated in a moist chamber (25 ± 2 °C, RH 85%) (Mahadevakumar et al. 2018). Typical disease symptoms were apparent on leaves, stems, rhizomes and plants at 4, 6, 5 and 15 days post inoculation, respectively. Fulfilling Koch's postulates, the fungal pathogens were isolated and purified from the inoculated site and were reconfirmed as S. delphinii based on the morphological features. To the best of our knowledge, this is the first report of S. delphinii causing southern blight on P. sibiricum in China. S. delphinii has a wide host range worldwide and often causes crop yield reduction. This study will be helpful for the prevention and control of P. sibiricum southern blight in the future.